Plants having modified growth characteristics and method for making the same

ABSTRACT

The present invention concerns a method for modifying the growth characteristics of plants by modulating expression in a plant of a nucleic acid sequence encoding a TAD protein and/or modulating activity in a plant of a TAD protein. The invention also relates to transgenic plants having modified growth characteristics, which plants have modulated expression of a nucleic acid encoding a TAD protein fragment.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 10/551,228(U.S. Patent Application Publication No. US-2006-0253932-A1), filed Sep.28, 2005 (allowed), which is a U.S. national phase of InternationalApplication PCT/EP2004/050136, filed Feb. 16, 2004, which designated theU.S. and claims priority of EP 03075975.7, filed Apr. 1, 2003, theentire contents of each of which is hereby incorporated by reference inthis application.

FIELD OF THE INVENTION

The present invention concerns a method for modifying plant growthcharacteristics. More specifically, the present invention concerns amethod for increasing yield by modulating expression of a nucleic acidsequence encoding a TAD protein and/or activity of a TAD protein in aplant. The present invention also concerns plants having modulatedexpression of a nucleic acid sequence encoding a TAD protein and/ormodulated activity of a TAD protein, which plants have increase yieldrelative to corresponding wild type plants.

BACKGROUND OF THE INVENTION

The AAA protein family (ATPases Associated with various cellularActivities, Kunau et al., Biochimie 75, 209-224, 1993) represents alarge group of proteins that all share a highly conserved ATP bindingdomain of about 230 amino acids, exhibiting ATPase activity (Neuwald etal., Genome Research 9, 27-43, 1999; Vale, J. Cell Biol. 150, F13-F19,2000). AAA proteins are widespread and have been characterised inArchaea, Eubacteria and all eukaryotic kingdoms. The AAA domains arerequired for protein functioning and are organised in hexameric ringsthat undergo conformational changes upon hydrolysis of ATP. This changein conformation, which is dependent on ATP hydrolysis, puts tension onbound proteins, and this mechanical activity allows unfolding ofassociated proteins, protein-protein dissociation etc. As a result, theAAA proteins play a role in different cellular processes, including cellcycle, organelle synthesis, mitochondrial functioning, vesicletransport, protein turnover, regulation of the cytoskeleton andintracellular motility. Thus, AAA proteins may represent a broad classof mechanoenzymes that have evolved unique ways of using a fundamentallysimilar conformational change in many different biological settings, butthe basis of interaction with their target proteins is still a matter ofspeculation (Vale, 2000). So far, most efforts of research are focusedon resolving the molecular structure and function of AAA ATPases, andconsequently nothing is known about their role on a macroscopic level,such as plant growth or yield.

Given the ever-increasing world population, it remains a major goal ofagricultural research to improve the efficiency of agriculture and toincrease the diversity of plants in horticulture. Conventional means forcrop and horticultural improvements utilise selective breedingtechniques to identify plants having desirable characteristics. However,such selective breeding techniques have several drawbacks, namely thatthese techniques are typically labour intensive and result in plantsthat often contain heterogeneous genetic complements that may not alwaysresult in the desirable trait being passed on from parent plants.Advances in molecular biology have allowed mankind to manipulate thegermplasm of animals and plants. Genetic engineering of plants entailsthe isolation and manipulation of genetic material (typically in theform of DNA or RNA) and the subsequent introduction of that geneticmaterial into a plant. Such technology has led to the development ofplants having various improved economic, agronomic or horticulturaltraits. A trait of particular economic interest is high yield.

DETAILED DESCRIPTION

Surprisingly, the inventors have found that modulating expression in aplant of a nucleic acid sequence encoding the ATPase domain derived froma TOB3 like protein (hereafter named TOB3 ATPase Domain, TAD) resultedin increased yield when compared to corresponding wild type plants.

Therefore according to a first embodiment of the present invention thereis provided a method for increasing yield of a plant compared tocorresponding wild type plants, comprising modulating expression in aplant of an isolated nucleic acid sequence encoding a TAD protein, or ahomologue, derivative or active fragment thereof and/or modulatingactivity of a TAD, a homologue, derivative or active fragment thereof.

The term TAD encoding nucleic acid/gene, as defined herein, refers toany nucleic acid encoding an ATPase domain derived from a TOB3 likeprotein, or the complement thereof. The nucleic acid may be derived(either directly or indirectly (if subsequently modified)) from anysource provided that the nucleic acid, when expressed in a plant, leadsto modulated expression of a TAD nucleic acid/gene. The nucleic acid maybe isolated from a microbial source, such as bacteria, archaea, yeast orfungi, or from a plant, algal or animal source. This nucleic acid may besubstantially modified from its native form in composition and/orgenomic environment through deliberate human manipulation. The nucleicacid molecule is preferably a homologous nucleic acid molecule, i.e. astructurally and/or functionally related nucleic acid molecule,preferably obtained from a plant, whether from the same plant species ordifferent. The nucleic acid molecule may be isolated from adicotyledonous species, preferably from the family Solanaceae, furtherpreferably from Nicotiana tabacum. More preferably, the nucleic acid isas represented by SEQ ID NO: 1 or a portion thereof or is a nucleic acidmolecule capable of hybridising therewith, which hybridising moleculesencode proteins having TAD (ATP binding and/or ATP hydrolysing)activity, i.e. similar biological activity to that of SEQ ID NO: 1; orthe nucleic acid encodes an amino acid represented by SEQ ID NO: 2 orencodes a homologue, derivative or active fragment thereof. The term TADencoding nucleic acid/gene also encompasses variants of the nucleic acidencoding a TAD due to the degeneracy of the genetic code; allelicvariants of the nucleic acid encoding a TAD; different splice variantsof the nucleic acid encoding a TAD and variants that are interrupted byone or more intervening sequences.

The term TAD protein, as defined herein, refers to proteins comprisingan ATPase domain derived from any TOB3 like protein. Preferably the TADis from Nicotiana tabacum, more preferably the TAD protein is a proteinas represented by SEQ ID NO: 2, or is a homologue, derivative or activefragment thereof, which homologues, derivatives or active fragments havesimilar biological activity to that of SEQ ID NO: 2. Methods formeasuring binding of ATP or for measuring ATPase activity are well knownin the art.

Advantageously, the method according to the present invention may alsobe practised using portions of a sequence represented by SEQ ID NO: 1 orby using sequences that hybridise (preferably under stringentconditions) to SEQ ID NO: 1, which hybridising sequences perform thesame biological function as SEQ ID NO 1 (that is: encode proteins havingTAD activity), or by using nucleic acids encoding homologues,derivatives or active fragments of a sequence according to SEQ ID NO 2.

Homologues of SEQ ID NO 2 may be found in various prokaryotic andeukaryotic organisms. The closest homologues however are generally foundin the plant kingdom. Suitable homologues of SEQ ID NO: 2 include otherTAD domains present in proteins such as represented in GenBankAccessions NP_(—)565435, NP_(—)195376, AAL87170, BAD08048, NP_(—)186956,BAD07862, NP_(—)197195 or PIR accessions A84563 or T51548.

Methods for the search and identification of TAD homologues would bewell within the realm of persons skilled in the art. Such methodscomprise comparison of the sequences represented by SEQ ID NO 1 or 2 ina computer readable format with sequences that are available in publicdatabases such as MIPS (http://mips.gsf.de/), GenBank(http://www.ncbi.nlm.nih.gov/Genbank/index.html) or EMBL NucleotideSequence Database (http://www.ebi.ac.uk/embl/index.html), usingalgorithms well known in the art for alignment or comparison ofsequences, such as GAP (Needleman and Wunsch, J. Mol. Biol. 48, 443-453(1970)), BESTFIT (using the local homology algorithm of Smith andWaterman (Advances in Applied Mathematics 2, 482-489 (1981))), BLAST(Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J., J.Mol. Biol. 215, 403-410 (1990)), FASTA and TFASTA (W. R. Pearson and D.J. Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988)). Thesoftware for performing BLAST analysis is publicly available at theNational Centre for Biotechnology Information.

These above-mentioned analyses for sequence homology can be done with afull-length query sequence or with certain regions of such a sequence,for example with conserved domains. Also the identification of familymembers of the TAD (as defined below) or the determination of thepercentage of sequence identity between the TAD and a homologue (asdefined below) can be performed by using these conserved sequences. Theidentification of such domains in a protein sequence would also be wellwithin the realm of the person skilled in the art and involve a computerreadable format of the nucleic acids used in the present invention, theuse of alignment software programs and the use of publicly availableinformation on protein domains, conserved motifs and boxes. Anintegrated search can be done using the INTERPRO database (Mulder etal., (2003) Nucl. Acids Res. 31, 315-318,http://www.ebi.ac.uk/interpro/scan.html) which combines severaldatabases on protein families, domains and functional sites, such as thePRODOM (Servant et al., (2002) Briefings in Bioinformatics 3, 246-251,http://prodes.toulouse.inra.fr/prodom/2002.1/html/home.php), PIR (Huanget al. (2003) Nucl. Acids Res. 31, 390-392, http://pir.georgetown.edu/)or Pfam (Bateman et al. (2002) Nucl. Acids Res. 30, 276-280,http://pfam.wustl.edu/) databases. Sequence analysis programs designedfor motif searching can be used for identification of conservedfragments, regions and domains as mentioned above. Suitable computerprograms to this end include for example MEME (Bailey and Elkan (1994)Proceedings of the Second International Conference on IntelligentSystems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park,Calif., http://meme.sdsc.edu/meme/website/intro.html).

The term TAD includes proteins homologous to a protein as presented inSEQ ID NO 2. “Homologues” of a TAD protein encompass peptides,oligopeptides, polypeptides, proteins and enzymes having amino acidsubstitutions, deletions and/or insertions relative to the unmodifiedprotein in question and having similar biological and functionalactivity as the unmodified protein from which they are derived. Toproduce such homologues, amino acids of the protein may be replaced byother amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or β-sheet structures). Conservativesubstitution tables are well known in the art (see for example Creighton(1984) Proteins. W.H. Freeman and Company).

When using an alignment program such as GAP, with a gap penalty of 10,an extend penalty of 0.5 and the BLOSUM 62 matrix, the homologues usefulin the methods according to the invention have at least 70% sequenceidentity with SEQ ID NO 2 in that part of their protein sequence thatcorresponds to SEQ ID NO 2. Typically, the homologues have at least 80%sequence identity or similarity to SEQ ID NO 2, preferably at least 85%sequence identity or similarity, further preferably at least 90%sequence identity or similarity to SEQ ID NO 2, most preferably at least95%, 96%, 97%, 98% or 99% sequence identity or similarity to SEQ ID NO2. The percentage of similarity can also be calculated using alignmentprograms such as GAP.

The homologues useful in the method according to the invention canalternatively be defined as having ATP binding activity and/or ATPaseactivity, and comprising a sequence of 22 consecutive amino acidresidues, which sequence is at least 90% identical to a correspondingsequence in SEQ ID NO 2.

Homologous proteins can be grouped in “protein families”. A proteinfamily can be defined by functional and sequence similarity analysis,using programs such as, for example, Clustal W. A neighbour-joining treeof proteins homologous to SEQ ID NO 2, generated by the Clustal Wprogram gives a good overview of their structural and ancestralrelationships. Advantageously also these family members are useful inthe methods of the present invention.

Two special forms of homology, orthologous and paralogous homology, areevolutionary concepts used to describe ancestral relationships of genes.The term “paralogous” relates to homologous genes that result from oneor more gene duplications within the genome of a species. The term“orthologous” relates to homologous genes in different organisms due toancestral relationship of these genes. The term “homologues” as usedherein also encompasses paralogues and orthologues of the proteinsuseful in the methods according to the invention. Orthologous genes canbe identified by querying one or more gene databases with a query geneof interest, using for example the BLAST program. The highest-rankingsubject genes that result from the search are then again subjected to aBLAST analysis, and only those subject genes that match again with thequery gene are retained as true orthologous genes. For example, to finda rice orthologue of an Arabidopsis thaliana gene, one may perform aBLASTN or TBLASTX analysis on a rice database (such as (but not limitedto) the Oryza sativa Nipponbare database available at the NCBI(http://www.ncbi.nlm.nih.gov) or the genomic sequences of rice(cultivars indica or japonica)). In a next step, the obtained ricesequences are used in a reverse BLAST analysis using an Arabidopsisdatabase. The results may be further refined when the resultingsequences are analysed with ClustalW and visualised in a neighbourjoining tree. The method can be used to identify orthologues from manydifferent species.

“Substitutional variants” of a protein are those in which at least oneresidue in an amino acid sequence has been removed and a differentresidue inserted in its place. Amino acid substitutions are typically ofsingle residues, but may be clustered depending upon functionalconstraints placed upon the polypeptide. Preferably, amino acidsubstitutions comprise conservative amino acid substitutions.“Insertional variants” of a protein are those in which one or more aminoacid residues are introduced into a predetermined site in a protein.Insertions can comprise amino-terminal and/or carboxy-terminal fusionsas well as intra-sequence insertions of single or multiple amino acids.Generally, insertions within the amino acid sequence will be smallerthan amino- or carboxy-terminal fusions, of the order of about 1 to 10residues. Examples of amino- or carboxy-terminal fusion proteins orpeptides include the binding domain or activation domain of atranscriptional activator as used in the yeast two-hybrid system, phagecoat proteins, (histidine)₆-tag, glutathione S-transferase-tag, proteinA, maltose-binding protein, dihydrofolate reductase, Tag•100 epitope,c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HAepitope, protein C epitope and VSV epitope. “Deletion variants” of aprotein are characterised by the removal of one or more amino acids fromthe protein, deletions will generally range from about 1 to 20 residues.Amino acid variants of a protein may readily be made using peptidesynthetic techniques well known in the art, such as solid phase peptidesynthesis and the like, or by recombinant DNA manipulations. Methods forthe manipulation of DNA sequences to produce substitution, insertion ordeletion variants of a protein are well known in the art. For example,techniques for making substitution mutations at predetermined sites inDNA are well known to those skilled in the art and include M13mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio),QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.),PCR-mediated site-directed mutagenesis or other site-directedmutagenesis protocols.

The term “derivatives” refers to peptides, oligopeptides, polypeptides,proteins and enzymes which may comprise substitutions, deletions oradditions of naturally and non-naturally occurring amino acid residuescompared to the amino acid sequence of a naturally-occurring form of theprotein, for example, as presented in SEQ ID NO: 2. “Derivatives” of aTAD encompass peptides, oligopeptides, polypeptides, proteins andenzymes which may comprise naturally occurring altered, glycosylated,acylated or non-naturally occurring amino acid residues compared to theamino acid sequence of a naturally-occurring form of the polypeptide. Aderivative may also comprise one or more non-amino acid substituentscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence to facilitate itsdetection, and non-naturally occurring amino acid residues relative tothe amino acid sequence of a naturally-occurring protein. “Activefragments” of a TAD protein encompass at least five contiguous aminoacid residues of a protein, which residues retain similar biologicaland/or functional activity to the naturally occurring protein.

Advantageously, the methods according to the present invention may alsobe practised using portions of a DNA or nucleic acid molecule, whichportions retain TAD activity, i.e. a similar biological function to thatof SEQ ID NO: 2. Portions of a DNA molecule refer to a piece of DNAderived or prepared from an original (larger) DNA molecule, which DNAportion, when expressed in a plant, gives rise to plants having modifiedgrowth characteristics. The portion may comprise many genes, with orwithout additional control elements, or may contain just spacersequences.

The present invention also encompasses nucleic acid molecules capable ofhybridising with a nucleic acid molecule encoding a TAD protein, whichnucleic acid molecules may also be useful in practising the methodsaccording to the invention. The term “hybridisation” as defined hereinis a process wherein substantially homologous complementary nucleotidesequences anneal to each other. The hybridisation process can occurentirely in solution, i.e. both complementary nucleic acids are insolution. Tools in molecular biology relying on such a process includethe polymerase chain reaction (PCR; and all methods based thereon),subtractive hybridisation, random primer extension, nuclease S1 mapping,primer extension, reverse transcription, cDNA synthesis, differentialdisplay of RNAs, and DNA sequence determination. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. Tools in molecular biology relying on such a processinclude the isolation of poly (A⁺) mRNA. The hybridisation process canfurthermore occur with one of the complementary nucleic acidsimmobilised to a solid support such as a nitro-cellulose or nylonmembrane or immobilised by e.g. photolithography to, for example, asiliceous glass support (the latter known as nucleic acid arrays ormicroarrays or as nucleic acid chips). Tools in molecular biologyrelying on such a process include RNA and DNA gel blot analysis, colonyhybridisation, plaque hybridisation, in situ hybridisation and microarray hybridisation. In order to allow hybridisation to occur, thenucleic acid molecules are generally thermally or chemically denaturedto melt a double strand into two single strands and/or to removehairpins or other secondary structures from single stranded nucleicacids. The stringency of hybridisation is influenced by conditions suchas temperature, salt concentration and hybridisation buffer composition.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. High stringency conditions forhybridisation thus include high temperature and/or low saltconcentration (salts include NaCl and Na₃-citrate) but can also beinfluenced by the inclusion of formamide in the hybridisation bufferand/or lowering the concentration of compounds such as SDS (sodiumdodecyl sulphate) in the hybridisation buffer and/or exclusion ofcompounds such as dextran sulphate or polyethylene glycol (promotingmolecular crowding) from the hybridisation buffer. Sufficiently lowstringency hybridisation conditions are particularly preferred for theisolation of nucleic acids homologous to the DNA sequences of theinvention defined supra. Elements contributing to homology includeallelism, degeneration of the genetic code and differences in preferredcodon usage.

“Stringent hybridisation conditions” and “stringent hybridisation washconditions” in the context of nucleic acid hybridisation experimentssuch as Southern and Northern hybridisations are sequence dependent andare different under different environmental parameters. For example,longer sequences hybridise specifically at higher temperatures. TheT_(m) is the temperature under defined ionic strength and pH, at which50% of the target sequence hybridises to a perfectly matched probe.Specificity is typically the function of post-hybridisation washes.Critical factors of such washes include the ionic strength andtemperature of the final wash solution. Generally, stringent conditionsare selected to be about 50° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature under defined ionic strength and pH, atwhich 50% of the target sequence hybridises to a perfectly matchedprobe. The T_(m) is dependent upon the solution conditions and the basecomposition of the probe, and may be calculated using the followingequation:

T _(m)=79.8° C.+(18.5x log [Na⁺])+(58.4° C.x %[G+C])−(820x(#bp induplex)⁻¹)−(0.5x % formamide)

More preferred stringent conditions are when the temperature is 20° C.below T_(m), and the most preferred stringent conditions are when thetemperature is 10° C. below T_(m). Non-specific binding may also becontrolled using any one of a number of known techniques such as, forexample, blocking the membrane with protein containing solutions,additions of heterologous RNA, DNA, and SDS to the hybridisation buffer,and treatment with Rnase. Wash conditions are typically performed at orbelow hybridisation stringency. Generally, suitable stringent conditionsfor nucleic acid hybridisation assays or gene amplification detectionprocedures are as set forth above. More or less stringent conditions mayalso be selected.

For the purposes of defining the level of stringency, reference canconveniently be made to Sambrook et al. (2001) Molecular Cloning: alaboratory manual, 3^(rd) Edition Cold Spring Harbor Laboratory Press,CSH, New York or to Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989). An example of low stringency conditions is4-6×SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours. Depending on thesource and concentration of the nucleic acid involved in thehybridisation, alternative conditions of stringency may be employed suchas medium stringent conditions. Examples of medium stringent conditionsinclude 1-4×SSC/0.25% w/v SDS at ≧45° C. for 2-3 hours. An example ofhigh stringency conditions includes 0.1-1×SSC/0.1% w/v SDS at 60° C. for1-3 hours. The skilled artisan is aware of various parameters which maybe altered during hybridisation and washing and which will eithermaintain or change the stringency conditions. For example, anotherstringent hybridisation condition is hybridisation at 4×SSC at 65° C.,followed by a washing in 0.1×SSC, at 65° C. for about one hour.Alternatively, an exemplary stringent hybridisation condition is in 50%formamide, 4×SSC at 42° C. Still another example of stringent conditionsinclude hybridisation at 62° C. in 6×SSC, 0.05×BLOTTO and washing at2×SSC, 0.1% w/v SDS at 62° C.

The methods according to the present invention may also be practisedusing an alternative splice variant of a nucleic acid molecule encodinga TAD protein. The term “alternative splice variant” as used hereinencompasses variants of a nucleic acid molecule in which selectedintrons and/or exons have been excised, replaced or added. Such variantswill be ones in which the biological activity of the protein remainsunaffected, which can be achieved by selectively retaining functionalsegments of the protein. Such splice variants may be found in nature orcan be manmade. Methods for making such splice variants are well knownin the art. Thus the invention also encompasses methods for modifyingthe growth characteristics of plants, in particular for increasingyield, comprising modulating expression in a plant of an alternativesplice variant of a nucleic acid molecule encoding a TAD and/or bymodulating activity and/or levels of a TAD encoded by the alternativesplice variant. Preferably, the splice variant is a splice variant ofthe sequence represented by SEQ ID NO: 1.

Advantageously, the methods according to the present invention may alsobe practised using allelic variants of a nucleic acid molecule encodinga TAD, preferably an allelic variant of a sequence represented by SEQ IDNO: 1. Allelic variants exist in nature and encompassed within themethods of the present invention is the use of these natural alleles.Allelic variants are further defined as to comprise single nucleotidepolymorphisms (SNPs) as well as small insertion/deletion polymorphisms(INDELs; the size of INDELs is usually less than 100 bp). SNPs andINDELs form the largest set of sequence variants in naturally occurringpolymorphic strains of most organisms. They are helpful in mapping genesand discovery of genes and gene functions. They are furthermore helpfulin identification of genetic loci, e.g. plant genes, involved indetermining processes such as growth rate, plant size and plant yield,plant vigour, disease resistance, stress tolerance etc. Many techniquesare nowadays available to identify SNPs and/or INDELs including (i) PCRfollowed by denaturing high performance liquid chromatography (DHPLC;e.g. Cho et al. (1999) Nature Genet. 23, 203-207); (ii) constantdenaturant capillary electrophoresis (CDCE) combined with high-fidelityPCR (e.g. Li-Sucholeiki et al. (1999) Electrophoresis 20, 1224-1232);(iii) denaturing gradient gel electrophoresis (Fischer and Lerman (1983)Proc. Natl. Acad. Sci. USA 80, 1579-1583); (iv) matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS;e.g. Ross et al. (2000) Biotechniques 29, 620-629); (v) real-timefluorescence monitoring PCR assays (Tapp et al. (2000) Biotechniques 28,732-738); (vi) Acrydite™ gel technology (Kenney et al. (1998)Biotechniques 25, 516-521); (vii) cycle dideoxy fingerprinting (CddF;Langemeier et al. (1994) Biotechniques 17, 484-490); (viii)single-strand conformation polymorphism (SSCP) analysis (Vidal-Puig andMoller (1994) Biotechniques 17, 490-496) and (ix) mini-sequencing primerextension reaction (Syvanen (1999) Hum. Mutat. 13, 1-10). The techniqueof ‘Targeting Induced Local Lesions in Genomes’ (TILLING; McCallum etal. (2000) Nat. Biotechnol. 18, 455-457; Plant Physiol. 123, 439-442),which is a variant of (i) supra, can also be applied to rapidly identifyan altered gene in e.g. chemically mutagenized plant individuals showinginteresting phenotypes.

The use of these allelic variants in particular conventional breedingprogrammes, such as in marker-assisted breeding is also encompassed bythe present invention; this may be in addition to their use in themethods according to the present invention. Such breeding programmessometimes require the introduction of allelic variations in the plantsby mutagenic treatment of a plant. One suitable mutagenic method is EMSmutagenesis. Identification of allelic variants then may take place by,for example, PCR. This is followed by a selection step for selection ofsuperior allelic variants of the [name protein] sequence in question andwhich give rise to altered growth characteristics in a plant. Selectionis typically carried out by monitoring growth performance of plantscontaining different allelic variants of the sequence in question, forexample, different allelic variants of SEQ ID NO: 1. Monitoring growthperformance can be done in a greenhouse or in the field. Furtheroptional steps include crossing plants, in which the superior allelicvariant was identified, with another plant. This could be used, forexample, to make a combination of interesting phenotypic features.Therefore, as mutations in the TAD gene may occur naturally, they mayform the basis for selection of plants showing higher yield.

The methods according to the present invention may also be practised byintroducing into a plant at least a part of a (natural or artificial)chromosome (such as a Bacterial Artificial Chromosome (BAC)), whichchromosome contains at least a gene/nucleic acid molecule encoding a TAD(such as SEQ ID NO: 1), preferably together with one or more relatedgenes from the same species or from the same family of genes, and/ornucleic acid molecule(s) encoding regulatory proteins for TAD expressionand/or activity. The present invention thus also encompasses a methodfor modifying growth characteristics of plants, in particular forincreasing yield, by introducing into a plant a part of a chromosomecomprising at least a gene/nucleic acid encoding a TAD, whichgene/nucleic acid encoding a TAD is under control of a seed-preferredpromoter and which artificial chromosome preferably also comprises oneor more related genes from the same species, and/or nucleic acidsequence(s) encoding regulatory proteins for TAD expression and/oractivity.

According to a preferred aspect of the present invention, overexpression(or increase of expression) of a nucleic acid is envisaged compared tocorresponding wild type plants. Increasing or decreasing expression (ormodulating expression) of a nucleic acid encoding a TAD proteinencompasses altered expression of this gene in the whole organism or inspecific cells or tissues. Altered expression of a gene may be effected,for example by chemical means and/or recombinant means. Modulatingexpression of a TAD gene may be effected directly (i.e. through themodulation of expression of the concerned TAD encoding gene itself). Inthe direct approach, the modulated expression may result from alteredexpression levels of an endogenous TAD gene and/or may result fromaltered expression of a TAD encoding nucleic acid that was previouslyintroduced into a plant. Additionally or alternatively, the modulationof expression as mentioned above is effected in an indirect way, forexample as a result of decreased or increased levels and/or activity offactors that control the expression of a TAD gene. The alteredexpression is to be understood as altered when compared to expression ofa corresponding TAD protein in corresponding wild type plants.

Advantageously, modulation of expression of a nucleic acid encoding aTAD protein and/or modulation of activity and/or levels of the TADprotein itself may be effected by chemical means, i.e. by exogenousapplication of one or more compounds or elements capable of modulatingexpression of a TAD gene (which may be either an endogenous gene or atransgene introduced into a plant). The term “exogenous application”taken in its broadest context includes contacting or administeringcells, tissues, organs or organisms with a suitable compound or element.The compound may be applied to a plant in a suitable form for uptake(such as through application to the soil for uptake via the roots, or byapplying directly to the leaves, for example by spraying). Suitablecompounds or elements for exogenous application include TAD encodingnucleic acids and nucleic acids that hybridise therewith. Additionallyor alternatively, contacting or administering cells, tissues, organs ororganisms with an interacting protein or with an inhibitor or activatorof the gene provides another exogenous means for modulation ofexpression of a nucleic acid encoding a TAD. Modulation of expression ofa nucleic acid encoding a TAD protein may also be effected as a resultof altered levels of factors that directly or indirectly activate orinactivate a TAD protein.

Furthermore, plants, seeds or other plant material can be subjected totreatment with mutagenic substances. Chemical substances effectingmutagenesis comprise N-nitroso-N-ethylurea, ethylene imine, ethylmethanesulphonate or diethyl sulphate. As an alternative, ionisingradiation such as γ-rays or X-rays can equally well be used. Methods forintroducing mutations and testing the effect of mutations (such asmodified protein expression and/or modified protein activity) are knownin the art. Encompassed by mutagenesis are methods employing chemicalmutagens, as well as physical mutagens, such as radiation. Anycharacteristic of the TAD protein can be altered by mutagenesis. Forexample these mutations can be responsible for the altered control of aTAD encoding gene, resulting in the desired expression level orexpression pattern of the gene. In particular, these mutations canresult in overexpression of TAD that is confined mainly to the seed.Alternatively and/or additionally, the activity or substrate specificityof the protein can be modified, or the affinity for a cofactor can beadapted. According to a preferred aspect of the invention saidmutagenesis results in an increase in expression and/or activity and/orlevels of a TAD protein in the plant seed.

Additionally or alternatively, and according to a preferred embodimentof the present invention, modulation of expression of a nucleic acidencoding a TAD and/or modulation of activity and/or levels of the TADprotein itself may be effected by recombinant means. Such recombinantmeans may comprise a direct and/or indirect approach for modulation ofexpression of a nucleic acid.

For example, an indirect recombinant approach may comprise introductioninto a plant of a nucleic acid capable of modulating activity and/orlevels of the protein in question (a TAD protein) and/or capable ofmodulating expression of the gene in question (a gene encoding a TADprotein). Examples of such nucleic acids to be introduced into a plantare nucleic acids encoding transcription factors, activators, inhibitorsor other ligands that bind to the promoter of the TAD gene or thatinteract with the TAD protein. Methods to test these kinds ofinteraction and to isolate the nucleic acids encoding these interactorsare for example yeast one-hybrid or yeast two-hybrid screening. The TADgene or the TAD protein may be wild type, i.e. the native or endogenousnucleic acid or polypeptide. Alternatively, it may be a nucleic acidderived from the same or another species, which gene is introduced as atransgene, for example by transformation. This transgene may besubstantially modified from its native form in composition and/orgenomic environment through deliberate human manipulation. Alsoencompassed by an indirect approach for modulating expression of a TADgene is the inhibition or stimulation of regulatory sequences, or theprovision of new regulatory sequences, that drive expression of thenative gene encoding a TAD or the transgene encoding a TAD. Suchregulatory sequences may be introduced into a plant. For example, theregulatory sequence introduced into the plant is a promoter, capable ofdriving the expression of an endogenous TAD gene.

A direct and preferred approach for modulating expression of a TAD genecomprises introduction into a plant of a nucleic acid molecule encodinga TAD protein or a homologue, derivative or active fragment thereof. Thenucleic acid may be introduced into a plant by, for example,transformation.

A more preferred way comprises the introduction into a plant of a TADgene as presented in SEQ ID NO 1. A most preferred way comprises theintroduction into a plant of a TAD encoding gene coupled in sensedirection to a seed-preferred promoter.

Methods for obtaining enhanced or increased expression of genes or geneproducts are well documented in the art and include overexpressiondriven by a suitable promoter and the use of transcription enhancers ortranslation enhancers. The term overexpression as used herein means anyform of expression that is additional to the original wild-typeexpression level. Preferably the nucleic acid to be introduced into theplant and/or to be overexpressed, is in a sense direction with respectto the promoter to which it is operably linked. The nucleic acid to beoverexpressed preferably encodes a TAD protein, further preferably a TADprotein of plant origin. More preferably, the nucleic acid moleculeencoding the TAD protein is isolated from a dicotyledonous plant,preferably of the family Solanaceae, further preferably the sequence isisolated from Nicotiana tabacum. Most preferably the nucleic acidsequence is as represented by SEQ ID NO: 1 or a portion thereof, orencodes an amino acid sequence as represented by SEQ ID NO: 2 or encodesa homologue, derivative or active fragment thereof. However, it shouldbe noted that the applicability of the invention does not rest upon theuse of the nucleic acid represented by SEQ ID NO: 1, nor upon thenucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2,but that other nucleic acid molecules encoding homologues, derivativesor active fragments of SEQ ID NO: 2, or portions of SEQ ID NO: 1, orsequences hybridising with SEQ ID NO: 1 may be used in the methods ofthe present invention.

According to another aspect of the present invention, decreasedexpression of a nucleic acid sequence is envisaged. Modulating geneexpression (whether by a direct or indirect approach) encompassesaltered transcript levels of a gene. Altered transcript levels can besufficient to induce certain phenotypic effects, for example via themechanism of cosuppression. Here the overall effect of overexpression ofa transgene is that there is less activity in the cell of the proteinencoded by a native gene having homology to the introduced transgene.Other examples of decreasing expression are also well documented in theart and include, for example, downregulation of expression by anti-sensetechniques, co-suppression techniques, RNAi techniques, smallinterference RNAs (siRNAs), microRNA (miRNA), the use of ribozymes, etc.Therefore according to a particular aspect of the invention, there isprovided a method for modulating growth characteristics of plants,including technologies that are based on the synthesis of antisensetranscripts, complementary to the mRNA of a TAD gene fragment, or basedon RNA interference. Advantageously, the methods according to thepresent invention may also be practised by downregulation of a nucleicacid sequence encoding a TAD. Plants having modified growthcharacteristics may be obtained by expressing a nucleic acid sequenceencoding a TAD in either sense or antisense orientation. Techniques fordownregulation are well known in the art. The terms “gene silencing” or“downregulation” of expression, as used herein, refer to lowering levelsof gene expression and/or levels of active gene product and/or levels ofgene product activity. Such decreases in expression may be accomplishedby, for example, the addition of coding sequences or parts thereof in asense orientation (if it is desired to achieve co-suppression).Therefore, according to one aspect of the present invention, the growthof a plant may be modified by introducing into a plant an additionalcopy (in full or in part) of a TAD gene fragment already present in ahost plant. The additional gene will silence the endogenous gene, givingrise to a phenomenon known as co-suppression.

Genetic constructs aimed at silencing gene expression may comprise theTAD encoding nucleotide sequence, for example as represented by SEQ IDNO: 1 (or one or more portions thereof) in a sense and/or antisenseorientation relative to the promoter sequence. The sense or antisensecopies of at least part of the endogenous gene in the form of direct orinverted repeats may be utilised in the methods according to theinvention. The growth characteristics of plants may also be modified byintroducing into a plant at least part of an antisense version of thenucleotide sequence represented, for example, by SEQ ID NO: 1. It shouldbe clear that part of the nucleic acid (a portion) could achieve thedesired result. Anti-sense sequences derived from corresponding genes ofthe same plant species are preferred to anti-sense sequences derivedfrom homologous genes, whether from the same or other plant species.

Another method for downregulation of gene expression or gene silencingcomprises use of ribozymes, for example as described in Atkins et al.1994 (WO 94/00012), Lenee et al. 1995 (WO 95/03404), Lutziger et al.2000 (WO 00/00619), Prinsen et al. 1997 (WO 97/3865) and Scott et al.1997 (WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by gene silencingstrategies as described by, among others, Angell and Baulcombe 1998 (WO98/36083), Lowe et al. 1989 (WO 98/53083), Lederer et al. 1999 (WO99/15682) or Wang et al. 1999 (WO 99/53050). Expression of an endogenousgene may also be reduced if the endogenous gene contains a mutation.Such a mutant gene may be isolated and introduced into the same ordifferent plant species in order to obtain plants having modified growthcharacteristics.

Preferably the overexpression of a TAD protein is primarily effected inthe seed of a plant, more preferably in the seed endosperm.Alternatively, the overexpression is effected in plant seedlings.Advantageously, performance of the methods according to the presentinvention results in plants having a variety of modified growthcharacteristics, such modified growth characteristics including modifiedyield or biomass, relative to corresponding wild type plants.Preferably, the modified growth characteristics are improved growthcharacteristics and include increased yield or biomass, relative tocorresponding wild type plants.

By “yield” is meant the amount of harvested material per area ofproduction. The term “increased yield” encompasses an increase inbiomass in one or more parts of a plant relative to the biomass ofcorresponding wild-type plants. Depending on the crop, the harvestedpart of the plant can be a different part or tissue of the plant, suchas seed (e.g. rice, sorghum or corn when grown for seed); totalabove-ground biomass (e.g. corn, when used as silage, sugarcane), root(e.g. sugar beet), fruit (e.g. tomato), cotton fibres, or any other partof the plant which is of economic value. For example, the methods of thepresent invention are used to increase seed yield of rice and of corn,or also to increase yield of silage corn in terms of overall aboveground biomass and energy content. The increase in yield encompasses anincrease in seed yield, which includes an increase in the total biomassof the seed (total seed weight) and/or an increase in the number offilled seeds and/or an increase in the total seed number. The increasein yield is also reflected in an increase of the harvest index, which isexpressed as a ratio of the yield of harvestable parts, such as seeds,over the total biomass.

Therefore, there is provided a method for increasing yield of a plant,and in particular seed yield, comprising introducing and overexpressingprimarily in the seed of this plant a nucleic acid sequence encoding aTAD protein, a homologue, a derivative or an active fragment thereofcompared to corresponding wild type plants, and wherein the increase ofyield comprises at least one of increased total weight of seeds,increased number of filled seeds, or increased harvest index, eachrelative to corresponding wild type plants.

Yield is by its nature a complex parameter whereby total yield dependson a number of yield components. The parameters for increased yield of acrop are well known by a person skilled in the art. By way of example,key yield components for corn include number of plants per hectare oracre, number of ears per plant, number of rows (of seeds) per ear,number of kernels per row, and Thousand Kernel Weight. The improvementin yield as obtained in accordance to the method of the invention, canbe obtained as a result in one or more of these yield components. By wayof example, key yield components for rice include number of plants perhectare or acre, number of panicles per plant, number of spikelets perpanicle, seed filling rate and thousand kernel weight. The improvementin yield as obtained in accordance to the method of the invention can beobtained as a result in one or more of these yield components,preferentially the improvement in yield is obtained primarily on thebasis of an increased number of flowers per panicle and an increasedseed filling rate.

According to a preferred feature of the present invention, performanceof the methods according to the present invention result in plantshaving modified yield. Preferably, the modified yield is increased seedyield and includes at least an increase in any one or more of number ofpanicles, number of spikelets per panicle, total seed number, number offilled seeds, total seed weight, Thousand Kernel Weight and harvestindex, each relative to control plants. Therefore, according to thepresent invention, there is provided a method for increasing totalweight of seeds, number of filled seeds and/or harvest index of plants,which method comprises modulating expression of a nucleic acid moleculeencoding a TAD protein and/or modulating activity of the TAD itself in aplant in a seed preferred way, preferably wherein the TAD protein isencoded by a nucleic acid sequence represented by SEQ ID NO: 1 or aportion thereof or by sequences capable of hybridising therewith orwherein the TAD is represented by SEQ ID NO: 2 or a homologue,derivative or active fragment thereof.

According to a further embodiment of the present invention, geneticconstructs and vectors to facilitate introduction and/or expression ofthe nucleotide sequences useful in the methods according to theinvention are provided. Therefore, according to the second embodiment ofthe present invention, there is provided a gene construct comprising:

-   -   a. a nucleic acid sequence capable of modulating expression of a        nucleic acid encoding a TAD protein and/or activity of a TAD        protein;    -   b. one or more control sequences capable of driving expression        of the nucleic acid sequence of (a)    -   c. a transcription termination sequence.

Constructs useful in the methods according to the present invention maybe created using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The genetic construct can be an expression vectorwherein the nucleic acid molecule is operably linked to one or morecontrol sequences allowing expression in prokaryotic and/or eukaryotichost cells.

According to a preferred embodiment of the invention, the geneticconstruct is an expression vector designed to overexpress the nucleicacid molecule; in particular overexpression primarily obtained in theseed, more particular in the endosperm of the plant seed is aimed at.Additionally and/or alternatively, the expression vector is designed tooverexpress the nucleic acid at the seedling stage. The nucleic acidmolecule capable of modulating expression of a nucleic acid encoding aTAD protein is preferably a nucleic acid molecule encoding a TAD or ahomologue, derivative or active fragment thereof, such as any of thenucleic acid molecules described hereinbefore. A preferred nucleic acidmolecule is the sequence represented by SEQ ID NO: 1 or a portionthereof or sequences capable of hybridising therewith or a nucleic acidmolecule encoding a sequence represented by SEQ ID NO: 2 or encoding ahomologue, derivative or active fragment thereof. Preferably, thisnucleic acid is cloned in sense orientation relative to the controlsequence to which it is operably linked.

Plants are transformed with a vector comprising the sequence of interest(i.e., the nucleic acid molecule encoding a TAD protein), which sequenceis operably linked to one or more control sequences (at least apromoter). The terms “regulatory element”, “regulatory sequence”,“control sequence” and “promoter” are all used herein interchangeablyand are to be taken in a broad context to refer to regulatory nucleicacid sequences capable of effecting expression of the sequences to whichthey are ligated. Encompassed by the aforementioned terms aretranscriptional regulatory sequences derived from a classical eukaryoticgenomic gene (including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence) andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner.Also included within the term is a transcriptional regulatory sequenceof a classical prokaryotic gene, in which case it may include a −35 boxsequence and/or −10 box transcriptional regulatory sequences. The term“regulatory element” also encompasses a synthetic fusion molecule orderivative which confers, activates or enhances expression of a nucleicacid molecule in a cell, tissue or organ. The term “operably linked” asused herein refers to a functional linkage between the promoter sequenceand the gene of interest, such that the promoter sequence is able toinitiate transcription of the gene of interest.

In order to obtain desired modified growth characteristics, it isimportant that the gene of interest is expressed at a suitable level andin a spatially and developmentally suitable pattern. Preferably, thenucleic acid molecule encoding a TAD protein is operably linked to aseed-preferred promoter. The term “seed preferred” promoter as definedherein refers to a promoter that is expressed predominantly in one ormore seed tissue(s). Preferably the seed-preferred promoter is anendosperm preferred promoter, more preferably a promoter as presented inGenBank under accession number X65064 (sequence from nucleotide 1 to672, hereafter named PRO0090), or a promoter of similar strength and/ora similar expression pattern. Therefore, the invention also provides amethod for modifying the growth characteristics of a plant, inparticular yield, comprising increasing expression in a plant of anucleic acid encoding a TAD protein, wherein the increased expression isprimarily obtained in the seed. Preferably, this expression is effectedunder control of a seed preferred promoter, more preferably the seedpreferred promoter is an endosperm preferred promoter, most preferablythe seed preferred promoter is PRO0090. It should be noted however thatthis PRO0090 promoter is also active in seedlings, hence the methods ofthe invention can also be practised with the use of a seedling preferredpromoter. Therefore, the invention also provides a method for modifyingthe growth characteristics of a plant, in particular yield, comprisingincreasing expression in a plant of a nucleic acid encoding a TADprotein, wherein the increased expression is primarily obtained in theseedling.

Promoter strength and/or expression pattern can be analysed for exampleby coupling the promoter to a reporter gene and assay the expression ofthe reporter gene in various tissues of the plant. One suitable reportergene well known to a person skilled in the art is beta-glucuronidase.The promoter strength and/or expression pattern can then be compared tothat of a well-characterised reference promoter, such as CaMV 35Spromoter or the seed preferred rice prolamin NRP33 promoter. A nonlimiting list of examples of other seed preferred promoters arepresented in Table 1, these promoters or derivatives thereof may also beuseful in the methods of the present invention.

TABLE 1 EXEMPLARY SEED-PREFERED PROMOTERS FOR USE IN THE PERFORMANCE OFTHE PRESENT INVENTION EXPRESSION GENE SOURCE PATTERN REFERENCEseed-specific genes seed Simon, et al., Plant Mol. Biol. 5: 191, 1985;Scofield, et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin seed Pearson, et al.,Plant Mol. Biol. 18: 235-245, 1992. legumin seed Ellis, et al., PlantMol. Biol. 10: 203-214, 1988. glutelin (rice) seed Takaiwa, et al., Mol.Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47,1987. zein seed Matzke et al Plant Mol Biol, 14(3): 323-32 1990 napAseed Stalberg, et al, Planta 199: 515- 519, 1996. wheat LMW and HMWendosperm Mol Gen Genet 216: 81-90, glutenin-1 1989; NAR 17: 461-2, 1989wheat SPA seed Albani et al, Plant Cell, 9: 171- 184, 1997 wheat α, β,γ-gliadins endosperm EMBO 3: 1409-15, 1984 barley ltr1 promoterendosperm barley B1, C, D, hordein endosperm Theor Appl Gen 98: 1253-62,1999; Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996 barleyDOF endosperm Mena et al, The Plant Journal, 116(1): 53-62, 1998 blz2endosperm EP99106056.7 synthetic promoter endosperm Vicente-Carbajosa etal., Plant J. 73: 629-640, 1998. rice prolamin NRP33 endosperm Wu et al,Plant Cell Physiology 39(8) 885-889, 1998 rice α-globulin Glb-1endosperm Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 rice OSH1embryo Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996 riceα-globulin REB/OHP-1 endosperm Nakase et al. Plant Mol. Biol. 33:513-522, 1997 rice ADP-glucose PP endosperm Trans Res 6: 157-68, 1997maize ESR gene family endosperm Plant J 12: 235-46, 1997 sorgumγ-kafirin endosperm PMB 32: 1029-35, 1996 KNOX embryo Postma-Haarsma etal, Plant Mol. Biol. 39: 257-71, 1999 rice oleosin embryo and Wu et at,J. Biochem., 123: 386, aleuron 1998 sunflower oleosin seed (embryo andCummins, et al., Plant Mol. Biol. dry seed) 19: 873-876, 1992 putativerice 40S ribosomal weak in protein endosperm rice alpha-globulin strongin endosperm rice alanine weak in aminotransferase endosperm trypsininhibitor ITR1 weak in (barley) endosperm rice WSI18 embryo + stressrice RAB21 embryo + stress rice oleosin 18 kd aleurone + embryo

Optionally, one or more terminator sequences may also be used in theconstruct introduced into a plant. The term “terminator” encompasses acontrol sequence which is a DNA sequence at the end of a transcriptionalunit which signals 3′ processing and polyadenylation of a primarytranscript and termination of transcription. Additional regulatoryelements may include transcriptional as well as translational enhancers.Those skilled in the art will be aware of terminator and enhancersequences which may be suitable for use in performing the invention.Such sequences would be known or may readily be obtained by a personskilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence which is required for maintenance and/orreplication in a specific cell type. One example is when a geneticconstruct is required to be maintained in a bacterial cell as anepisomal genetic element (e.g. plasmid or cosmid molecule). Preferredorigins of replication include, but are not limited to, the f1-ori andcolE1.

The genetic construct may optionally comprise a selectable marker gene.As used herein, the term “selectable marker gene” includes any genewhich confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a nucleic acid construct of theinvention. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker proteinsinclude proteins conferring resistance to antibiotics (such as nptIIthat phosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin), to herbicides (for example bar which provides resistance toBasta; aroA or gox providing resistance against glyphosate), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source). Visual marker genes result in theformation of colour (for example β-glucuronidase, GUS), luminescence(such as luciferase) or fluorescence (Green Fluorescent Protein, GFP,and derivatives thereof).

In a preferred embodiment, the genetic construct as mentioned above,comprises a TAD in sense orientation coupled to a promoter that ispreferably a seed-preferred and/or seedling preferred promoter, such asfor example the PRO0090 promoter. Therefore, another aspect of thepresent invention is a vector construct comprising an expressioncassette essentially similar to SEQ ID NO 3, comprising the PRO0090promoter, the tobacco TAD gene and the T-zein+T-rubisco transcriptionterminator sequence. A sequence essentially similar to SEQ ID NO 3encompasses a first nucleic acid sequence encoding a protein homologousto SEQ ID NO 2 or hybridising to SEQ ID NO 1, which first nucleic acidis operably linked to the PRO0090 promoter, or a promoter with a similarexpression pattern and level, and which first nucleic acid is optionallylinked to a transcription termination sequence.

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants obtainable by the methods according to the presentinvention, which plants have increased yield, particularly, increasedtotal weight of seeds, increased total seed number, increased number offilled seeds, and/or increased harvest index, and which plants havemodulated expression of a nucleic acid encoding a TAD protein and/ormodulated activity of a TAD protein. Preferably, the modulatedexpression and/or activity is effected mainly in the seed and/or in theseedling when compared to corresponding wild type plants. Preferably,the modulated expression and/or activity is increased expression and/oractivity compared to corresponding wild type plants.

More specifically, the present invention provides a method for theproduction of transgenic plants having modified growth characteristics,which method comprises:

-   -   (i) introducing into a plant or into a plant cell a nucleic acid        molecule or a portion thereof encoding a TAD protein or a        homologue, derivative or active fragment thereof;    -   (ii) cultivating the plant cell under conditions promoting        regeneration and mature plant growth.

The protein itself and/or the nucleic acid itself may be introduceddirectly into a plant cell or into the plant itself (includingintroduction into a tissue, organ or any other part of the plant).According to a preferred feature of the present invention, the nucleicacid is preferably introduced into a plant by transformation. Thenucleic acid is preferably as represented by SEQ ID NO: 1 or a portionthereof or sequences capable of hybridising therewith, or is a nucleicacid encoding an amino acid sequence represented by SEQ ID NO: 2 or ahomologue, derivative or active fragment thereof. The nucleic acidsequence is preferably under control of a seed/seedling-preferredpromoter, more preferably the PRO0090 promoter.

The term “transformation” as referred to herein encompasses the transferof an exogenous polynucleotide into a host cell, irrespective of themethod used for transfer. Plant tissue capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with a genetic construct of the present invention and awhole plant regenerated therefrom. The particular tissue chosen willvary depending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristem, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Thepolynucleotide may be transiently or stably introduced into a host celland may be maintained non-integrated, for example, as a plasmid.Alternatively, it may be integrated into the host genome. The resultingtransformed plant cell can then be used to regenerate a transformedplant in a manner known to persons skilled in the art.

Transformation of a plant species is now a fairly routine technique.Advantageously, any of several transformation methods may be used tointroduce the gene of interest into a suitable ancestor cell.Transformation methods include the use of liposomes, electroporation,chemicals that increase free DNA uptake, injection of the DNA directlyinto the plant, particle gun bombardment, transformation using virusesor pollen and microprojection. Methods may be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1882, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol.8, 363-373); electroporation of protoplasts (Shillito R. D. et al., 1985Bio/Technol 3, 1099-1102); microinjection into plant material (CrosswayA. et al., 1986, Mol. Gen. Genet. 202, 179-185); DNA or RNA-coatedparticle bombardment (Klein T. M. et al., 1987, Nature 327, 70)infection with (non-integrative) viruses and the like. A preferredmethod for rice transformation according to the present invention is theprotocol of Hiei et al. (Plant J. 6, 271-282, 1994). Preferred methodsto transform corn with a high efficiency are the protocols described inIshida et al. (1996, Nat. Biotechnol. 14, 745-50), and in Frame et al.(2002, Plant Physiol. 129, 13-22), which disclosures are incorporated byreference herein as if fully set forth.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, bothtechniques being well known to persons having ordinary skill in the art.

In a next step of selection, transformed plants are evaluated for thedesired phenotypes. It is well known to persons skilled in the art, likeplant molecular biologists, that the expression of transgenes in plants,and hence also the phenotypic effect due to expression of suchtransgene, can differ among different independently obtained transgeniclines and progeny thereof. The transgenes present in differentindependently obtained transgenic plants differ from each other by thechromosomal insertion locus as well as by the number of transgene copiesinserted in that locus and the configuration of those transgene copiesin that locus. Differences in expression levels can be ascribed toinfluence from the chromosomal context of the transgene (the so-calledposition effect) or from silencing mechanisms triggered by certaintransgene configurations (e.g. inwards facing tandem insertions oftransgenes are prone to silencing at the transcriptional orpost-transcriptional level).

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant parts,seeds and propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedin the parent by the methods according to the invention. The inventionalso includes host cells containing an isolated nucleic acid moleculeencoding a TAD protein. Preferred host cells according to the inventionare plant cells. The invention also extends to harvestable parts of aplant according to the invention, such as, but not limited to, seeds,leaves, fruits, flowers, stems or stem cultures, rhizomes, roots, tubersand bulbs.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, fruits, flowers,shoots, leaves, stems, roots (including tubers), and plant cells,tissues and organs. The term “plant” also therefore encompassessuspension cultures, embryos, meristematic regions, callus tissue,gametophytes, sporophytes, pollen, and microspores. Plants that areparticularly useful in the methods of the invention include algae,ferns, and all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants, including fodderor forage legumes, ornamental plants, food crops, trees, or shrubsselected from the list comprising Abelmoschus spp., Acer spp., Actinidiaspp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus,Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp,Artocarpus spp., Asparagus officinalis, Avena sativa, Averrhoacarambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris,Brassica spp., Cadaba farinosa, Camellia sinensis, Canna indica,Capsicum spp., Carica papaya, Carissa macrocarpa, Carthamus tinctorius,Carya spp., Castanea spp., Cichorium endivia, Cinnamomum spp., Citrulluslanatus, Citrus spp., Cocos spp., Coffea spp., Cola spp., Colocasiaesculenta, Corylus spp., Crataegus spp., Cucumis spp., Cucurbita spp.,Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscoreaspp., Diospyros spp., Echinochloa spp., Eleusine coracana, Eriobotryajaponica, Eugenia uniflora, Fagopyrum spp., Fagus spp., Ficus carica,Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp., Gossypiumhirsutum, Helianthus spp., Hibiscus spp., Hordeum spp., Ipomoea batatas,Juglans spp., Lactuca sativa, Lathyrus spp., Lemna spp., Lens culinaris,Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula,Lupinus spp., Macrotyloma spp., Malpighia emarginata, Malus spp., Mammeaamericana, Mangifera indica, Manihot spp., Manilkara zapota, Medicagosativa, Melilotus spp., Mentha spp., Momordica spp., Morus nigra, Musaspp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryzaspp., Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Perseaspp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp.,Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopisspp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis,Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Rubusspp., Saccharum spp., Sambucus spp., Secale cereale, Sesamum spp.,Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tamarindusindica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticumspp., Vaccinium spp., Vicia spp., Vigna spp., Vitis spp., Zea mays,Zizania palustris, Ziziphus spp., amongst others.

The methods of the present invention are favourable to apply to cropplants because the methods of the present invention are used to increaseyield, in particular the seed yield, more particular total weight ofseeds, number of filled seeds and harvest index of a plant. Therefore,the methods of the present invention are particularly useful for cropplants cultivated for their seeds, such as cereals, sunflower, soybean,cotton, pea, flax, lupines, canola etc, but are also useful for cropsthat are cultivated for their biomass. According to a preferred featureof the present invention, the plant is a crop plant comprising soybean,sunflower, canola, alfalfa, rapeseed or cotton. Further preferably, theplant according to the present invention is a monocotyledonous plant,including members of the Poaceae, such as sugarcane, most preferably acereal, such as rice, maize, wheat, millet, barley and sorghum.Accordingly, a particular embodiment of the present invention relates toa method to increase yield, in particular total weight of seeds, numberof filled seeds, total seed number and/or harvest index of a cereal.

The present invention also relates to the use of an isolated nucleicacid encoding a TAD protein and to the use of portions thereof ornucleic acids hybridising therewith in modifying the growthcharacteristics of plants, preferably in increasing the yield of aplant, more preferably seed yield, in particular the total weight ofseeds, the number of filled seeds, total seed number and/or the harvestindex of a plant, and wherein the nucleic acid encoding a TAD protein,the portions thereof or the nucleic acids hybridising therewith areexpressed in a seed preferred manner. The present invention also relatesto use of a TAD protein and to the use of homologues, derivatives andactive fragments thereof in modifying the yield of plants, in particularthe use for increasing the total weight of seeds, the number of filledseeds, total seed number and/or the harvest index of plants. The nucleicacid sequence is preferably as represented by SEQ ID NO: 1 or a portionthereof or sequences capable of hybridising therewith or encodes anamino acid sequence represented by SEQ ID NO: 2 or a homologue,derivative or active fragment thereof. The invention encompasses alsothe use of plants or plant parts (including seeds) for processing, whichplants or plant parts have modulated expression of a nucleic acidencoding a TAD protein and/or modulated activity of a TAD protein. Suchuses for processing include for example the use in food or feedproduction, in brewing, in the production of industrial proteins orpharmaceuticals, in sugar or oil production.

The methods according to the present invention result in plants havingmodified growth characteristics, as described hereinbefore. Theseadvantageous growth characteristics may also be combined with othereconomically advantageous traits, such as further yield-enhancingtraits, tolerance to various stresses, traits modifying variousarchitectural features and/or biochemical and/or physiological features.

Accordingly, the methods of the present invention can also be used inso-called “gene stacking” procedures.

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1: Schematic presentation of the entry clone p69, containingCDS0671 within the AttL1 and AttL2 sites for Gateway® cloning in thepDONR201 backbone. CDS0671 is the internal code for the TOB3-likeAAA-ATPase domain coding sequence of Nicotiana tabacum BY2 cells. Thisvector contains also a bacterial kanamycine-resistance cassette and abacterial origin of replication.

FIG. 2: Binary vector for the expression in Oryza sativa of theNicotiana tabacum BY2 cells TOB3-like AAA-ATPase domain gene (CDS0671)under the control of the endosperm/seedling preferred promoter PRO0090.This vector contains a T-DNA derived from the Ti Plasmid, limited by aleft border (LB repeat, LB Ti C58) and a right border (RB repeat, RB TiC58)). From the left border to the right border, this T-DNA contains: aplant selectable marker and a screenable marker for selection andscreening of transformed plants; the PRO0090-CDS0671-zein andrbcS-deltaGA double terminator cassette for expression of the Nicotianatabacum BY2 cells TOB3-like AAA-ATPase domain gene. This vector alsocontains an origin of replication from pBR322 for bacterial replicationand a selectable marker (Spe/SmeR) for bacterial selection withspectinomycin and streptomycin.

FIG. 3: Sequence listing.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone.

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1984), Current Protocols in Molecular Biology, CurrentProtocols. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfase (1993) by R. D. D. Croy,published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications (UK).

Example 1 Cloning of CDS0671

Cloning of the TAD Gene Fragment from Tobacco

A cDNA-AFLP experiment was performed on a synchronized tobacco BY2 cellculture (Nicotiana tabacum L. cv. Bright Yellow-2), and BY2 expressedsequence tags that were cell cycle modulated were elected for furthercloning. The expressed sequence tags were used to screen a tobacco cDNAlibrary and to isolate the cDNA of interest, namely one coding forTOB3-like AAA-ATPase domain gene (CDS0671).

Synchronization of BY2 Cells.

A tobacco BY2 (Nicotiana tabacum L. cv. Bright Yellow-2) cultured cellsuspension was synchronized by blocking cells in early S-phase withaphidicolin as follows. The cell suspension of Nicotiana tabacum L. cv.Bright Yellow 2 was maintained as described (Nagata et al. Int. Rev.Cytol. 132, 1-30, 1992). For synchronization, a 7-day-old stationaryculture was diluted 10-fold in fresh medium supplemented withaphidicolin (Sigma-Aldrich, St. Louis, Mo.; 5 mg/l), a DNA-polymerase ainhibiting drug. After 24 h, cells were released from the block byseveral washings with fresh medium after which their cell cycleprogression resumed.

RNA Extraction and cDNA Synthesis.

Total RNA was prepared using LiCl precipitation (Sambrook et al, 2001)and poly(A⁺) RNA was extracted from 500 μg of total RNA using Oligotexcolumns (Qiagen, Hilden, Germany) according to the manufacturer'sinstructions. Starting from 1 μg of poly(A⁺) RNA, first-strand cDNA wassynthesized by reverse transcription with a biotinylated oligo-dT25primer (Genset, Paris, France) and Superscript II (Life Technologies,Gaithersburg, Md.). Second-strand synthesis was done by stranddisplacement with Escherichia coli ligase (Life Technologies), DNApolymerase I (USB, Cleveland, Ohio) and RNAse-H (USB).

cDNA-AFLP Analysis.

Five hundred ng double-stranded cDNA was used for AFLP analysis asdescribed (Vos et al., Nucleic Acids Res. 23 (21) 4407-4414, 1995;Bachem et al., Plant J. 9 (5) 745-53, 1996) with modifications. Therestriction enzymes used were BstYI and Msel (Biolabs) and the digestionwas done in two separate steps. After the first restriction digest withone of the enzymes, the 3′ end fragments were trapped on Dyna beads(Dynal, Oslo, Norway) by means of their biotinylated tail, while theother fragments were washed away. After digestion with the secondenzyme, the released restriction fragments were collected and used astemplates in the subsequent AFLP steps. For pre-amplifications, a Mselprimer without selective nucleotides was combined with a BstYI primercontaining either a T or a C as 3′ most nucleotide. PCR conditions wereas described (Vos et al., 1995). The obtained amplification mixtureswere diluted 600-fold and 5 μl was used for selective amplificationsusing a P33-labeled BstYI primer and the Amplitaq-Gold polymerase (RocheDiagnostics, Brussels, Belgium). Amplification products were separatedon 5% polyacrylamide gels using the Sequigel system (Biorad). Dried gelswere exposed to Kodak Biomax films as well as scanned in aPhosphorlmager (Amersham Pharmacia Biotech, Little Chalfont, UK).

Characterization of AFLP Fragments.

Bands corresponding to differentially expressed transcripts, among whichthe (partial) transcript corresponding to SEQ ID NO 1 (or CDS0671), wereisolated from the gel and eluted. DNA was reamplified under the sameconditions as for selective amplification. Sequence information wasobtained either by direct sequencing of the reamplified polymerase chainreaction product with the selective BstYI primer or after cloning thefragments in pGEM-T easy (Promega, Madison, Wis.) and sequencing ofindividual clones. The obtained sequences were compared againstnucleotide and protein sequences present in the publicly availabledatabases by BLAST sequence alignments (Altschul et al., Nucleic AcidsRes. 25 (17) 3389-3402 1997). When available, tag sequences werereplaced with longer EST or isolated cDNA sequences to increase thechance of finding significant homology. The physical cDNA clonecorresponding to SEQ ID NO 1 (CDS0671) was subsequently amplified from acommercial Tobacco cDNA library as follows.

Cloning of the TAD Gene Fragment (CDS0671)

A cDNA library with an average size of inserts of 1,400 bp was preparedfrom poly(A⁺) RNA isolated from actively dividing, non-synchronized BY2tobacco cells. These library-inserts were cloned in the vectorpCMVSPORT6.0, comprising an attB Gateway cassette (Life Technologies).From this library, 46,000 clones were selected, arrayed in 384-wellmicrotiter plates, and subsequently spotted in duplicate on nylonfilters. The arrayed clones were screened using pools of severalhundreds of radioactively labelled tags as probe (including the BY2-tagcorresponding to the sequence CDS0671, SEQ ID NO 1). Positive cloneswere isolated (among which was the clone corresponding to CDS0671, SEQ INO 1), sequenced, and aligned with the tag sequence. In cases wherehybridisation with the tag failed, the full-length cDNA corresponding tothe tag was selected by PCR amplification: tag-specific primers weredesigned using primer3 program(http://www-genome.wi.mit.edu/genome_software/other/primer3.html) andused in combination with a common vector primer to amplify partial cDNAinserts. Pools of DNA from 50,000, 100,000, 150,000, and 300,000 cDNAclones were used as templates in the PCR amplifications. Amplificationproducts were then isolated from agarose gels, cloned, sequenced andtheir sequence aligned with those of the tags. Next, the full-lengthcDNA corresponding to the nucleotide sequence of SEQ ID NO 1 was clonedfrom the pCMVsport6.0 library vector into pDONR201, a Gateway® donorvector (Invitrogen, Paisley, UK) via a LR reaction, resulting in theentry clone p69 (FIG. 1).

Example 2 Vector Construction for Transformation with PRO0090-CDS0671Cassette

The entry clone p69 was subsequently used in an LR reaction with p0830,a destination vector used for Oryza sativa transformation. This vectorcontains as functional elements within the T-DNA borders: a plantselectable marker; a screenable marker; and a Gateway cassette intendedfor LR in vivo recombination with the sequence of interest alreadycloned in the entry clone. Promoter PRO0090 was located upstream of thisGateway cassette.

After the LR recombination step, the resulting expression vector p74(FIG. 2) was transformed into Agrobacterium strain LBA4044 andsubsequently into Oryza sativa plants.

Example 3 Transformation of Rice with PRO0129 Up-CDS0716

Mature dry seeds of Oryza sativa japonica cultivar Nipponbare weredehusked. Sterilization was done by incubating the seeds for one minutein 70% ethanol, followed by 30 minutes in 0.2% HgCl₂ and by 6 washes of15 minutes with sterile distilled water. The sterile seeds were thengerminated on a medium containing 2,4-D (callus induction medium). Aftera 4-week incubation in the dark, embryogenic, scutellum-derived calliwere excised and propagated on the same medium. Two weeks later, thecalli were multiplied or propagated by subculture on the same medium foranother 2 weeks. 3 days before co-cultivation, embryogenic callus pieceswere sub-cultured on fresh medium to boost cell division activity. TheAgrobacterium strain LBA4404 harbouring the binary vector p3076 was usedfor co-cultivation. The Agrobacterium strain was cultured for 3 days at28° C. on AB medium with the appropriate antibiotics. The bacteria werethen collected and suspended in liquid co-cultivation medium at an OD₆₀₀of about 1. The suspension was transferred to a petri dish and the calliwere immersed in the suspension during 15 minutes. The callus tissueswere then blotted dry on a filter paper, transferred to solidifiedco-cultivation medium and incubated for 3 days in the dark at 25° C.

Co-cultivated callus was then grown on 2,4-D-containing medium for 4weeks in the dark at 28° C. in the presence of a selective agent at asuitable concentration. During this period, rapidly growing resistantcallus islands developed. Upon transfer of this material to aregeneration medium and incubation in the light, the embryogenicpotential was released and shoots developed in the next four to fiveweeks. Shoots were excised from the callus and incubated for 2 to 3weeks on an auxin-containing medium from which they were transferred tosoil. Hardened shoots were grown under high humidity and short days in agreenhouse. Finally seeds were harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges, 1996, Chan et al., 1993, Hiei et al.,1994).

Example 4 Evaluation of Transgenic Rice Transformed with PRO0129-CDS1585

Approximately 15 to 20 independent T0 rice transformants were generated.The primary transformants were transferred from tissue culture chambersto a greenhouse for growing and harvest of T1 seed. 7 events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring screenable marker expression.

The selected T1 plants were transferred to a greenhouse. Each plantreceived a unique barcode label to unambiguously link the phenotypingdata to the corresponding plant. The selected T1 plants were grown onsoil in 10 cm diameter pots under the following environmental settings:photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytimetemperature=28° C. or higher, night time temperature=22° C., relativehumidity=60-70%. Transgenic plants and the corresponding nullizygoteswere grown side-by-side at random positions. From the stage of sowinguntil the stage of maturity each plant was passed several times througha digital imaging cabinet. At each time point, digital images (2048×1536pixels, 16 million colours) were taken of each plant from at least 6different angles.

The mature primary panicles were harvested, bagged, barcode-labelled andthen dried for three days in an oven at 37° C. The panicles were thenthreshed and all the seeds collected. The filled husks were separatedfrom the empty ones using an air-blowing device. After separation, bothseed lots were then counted using a commercially available countingmachine. The empty husks were discarded. The filled husks were weighedon an analytical balance and the cross-sectional area of the seeds wasmeasured using digital imaging. This procedure resulted in the followingset of seed-related parameters:

-   -   (i) Number of filled seeds: was determined by counting the        number of filled husks that remained after the separation step.    -   (ii) Total seed weight per plant: the yield was measured by        weighing all filled husks harvested from a plant.    -   (iii) Harvest index of plants: the harvest index in the present        invention is defined as the ratio between the total seed weight        and the above ground area (mm²), multiplied by a factor 10⁶.

These parameters were derived in an automated way from the digitalimages using image analysis software and were analysed statistically. Atwo factor ANOVA (analyses of variance) corrected for the unbalanceddesign was used as statistical model for the overall evaluation of plantphenotypic characteristics. An F-test was carried out on all theparameters measured of all the plants of all the events transformed withthat gene. The F-test was carried out to check for an effect of the geneover all the transformation events and to verify for an overall effectof the gene, also named herein a “global gene effect”. A significantvalue for the F test shows there is a “gene” effect, meaning that it isnot only the presence or the position of the gene that is causing theeffect. The threshold for significance for a true global gene effect isset at 5% probability level for the F test.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “Null segregants” or “Nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at a 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

Vegetative growth and seed yield were measured according to the methodsas described above. It was found that the total weight of seeds, thenumber of filled seeds and the harvest index were increased in theplants transformed with the TAD coding sequence when compared thecontrol plants without the TAD coding sequence.

The data obtained in the first experiment were confirmed in a secondexperiment with T2 plants. Seed batches from the positive plants (bothhetero- and homozygotes) in T1, were screened by monitoring markerexpression. For each chosen event, the heterozygote seed batches werethen retained for T2 evaluation. Within each seed batch an equal numberof positive and negative plants were grown in the greenhouse forevaluation. Three lines that had the correct expression pattern wereselected for further analysis.

A total number of 120 TAD transformed plants were evaluated in the T2generation, that is 40 plants per event of which 20 were positive forthe transgene and 20 negative.

Example 5 Results of the Evaluation of Transgenic Plants Transformedwith PRO0090-CDS0671

In generation T1, 4 of the 5 tested lines showed an increase in thenumber of filled seeds, the total weight of seeds and in the harvestindex. For the best line (line 21), such increase amounted to around 50%for three different seed yield parameters, each with a low p-value(Table 2).

TABLE 2 increase of seed yield for line 21 Parameter % increase p-valueTotal seed weight 58 0.0611 Number of filled seeds 58 0.0403 HarvestIndex 47 0.0210

The mean increase calculated from the data of the four positive T1 linesand the three T2 lines that were taken for confirmation, versus thenullizygous plants for the different seed yield parameters is listed inTable 3.

TABLE 3 mean increase in seed yield in T1 and T2 generation T1, % T2, %p-value, T1 and T2 parameter increase increase combined Total seedweight 34 34 0.0058 Number of filled 37 27 0.0134 seeds Harvest Index 2524 0.0053

The results for the T1 plants were confirmed with T2 plants. Here too,there was an increase for the various parameters (Table 3). When thedata for the T1 and T2 plants were combined and re-analysed, thepositive effects for the number of filled seeds, the total weight ofseeds and the harvest index were found to be highly significant (seep-values in Table 3, right hand column).

Example 6 Use of the Invention in Corn

The invention described herein can also be used in maize. To this aim,the TAD gene, or the maize orthologue thereof is cloned under control ofa suitable promoter, preferably a seed and/or seedling preferredpromoter in a plant transformation vector suited forAgrobacterium-mediated corn transformation. Such vectors and methods forcorn transformation have been described in literature (EP0604662,EP0672752, EP0971578, EP0955371, EP0558676, Ishida et al. 1996; Frame etal., 2002). Transgenic plants made by these methods are grown in thegreenhouse for T1 seed production. Heritability is checked by progenysegregation analysis. Copy number of the transgene is checked byquantitative real-time PCR and/or Southern blot analysis. Expressionlevels of the transgene are determined by reverse PCR and/or Northernanalysis. Transgenic lines with single copy insertions of the transgeneand with varying levels of transgene expression are selected for T2 seedproduction through selfing or for crossing to different germplasm.Progeny seeds are germinated and grown in the field or in the greenhousein conditions well adapted for maize (16:8 hr photoperiod, 26-28° C.daytime and 22-24° C. night time temperature) as well underwater-deficient, nitrogen-deficient, and excess NaCl conditions. In thecase of selfing, null segregants from the same parental line, as well aswild type plants of the same cultivar are used as controls. In the caseof crossing, transgenics, null segregants and wild type plants of thesame cultivar are crossed to a chosen parent and F1 plants from thetransgenic cross are compared to F1 plants from the null segregant andthe wild type crosses. The progeny plants resulting from the selfing orthe crosses are evaluated on different biomass and growth parameters,including plant height, stem thickness, number of leaves, total aboveground area, leaf greenness, time to maturity, flowering time, earnumber, harvesting time. The seeds of these lines are also checked onvarious parameters, such as grain size, total grain yield per plant, andgrain quality (starch content, protein content and oil content). Linesthat are most significantly improved versus the controls for any of theabove-mentioned parameters are selected for further field testing andmarker-assisted breeding, with the objective of transferring thefield-validated transgenic traits into commercial germplasm. Methods fortesting maize for growth and yield-related parameters in the field arewell established in the art, as are techniques for introgressingspecific loci (such as transgene containing loci) from one germplasminto another.

1. Transgenic plant having increased yield compared to correspondingwild type plants, characterised in that said transgenic plant hasmodulated expression of a nucleic acid sequence encoding a TAD proteinand/or modulated activity of a TAD protein.
 2. Transgenic plant of claim1, wherein said modulated expression and/or modulated activity isincreased expression and/or increased activity, compared tocorresponding wild type plants.
 3. Transgenic plant according to claim1, wherein said plant is a crop plant such as soybean, sunflower,canola, alfalfa, rapeseed or cotton, preferably a monocotyledonous plantsuch as sugarcane, most preferably a cereal, such as rice, maize, wheat,millet, barley, sorghum.
 4. Transgenic plant cells, transgenic plantparts, including harvestable parts, propagules, seeds or progeny, of aplant according to claim
 1. 5. Use of a transgenic plant according toclaim 1, plant parts or seeds thereof for processing.
 6. A method forincreasing seed yield of a plant compared to corresponding controlplants comprising modulating expression in a plant of a nucleic acidsequence encoding a TAD protein and/or modulating activity of a TADprotein.
 7. Method for the production of a transgenic plant havingincreased seed yield compared to corresponding control plants, whichmethod comprises: a) introducing into a plant or plant cell a nucleicacid sequence encoding a polypeptide of SEQ ID NO:2; b) cultivating theplant or plant cell under conditions promoting regeneration and matureplant growth.